Stability Indicating HPLC Method Development for Estimation of Montelukast Sodium and Acebrophylline in Combined Dosage Form

ISSN No: 2321 – 8630, V – 1, I – 1, 2014 Journal Club for Pharmaceutical Sciences (JCPS)

Manuscript No: JCPS/RES/2014/17, Received On: 03/08/2014, Revised on: 09/08/2014, Accepted On: 13/08/2014

RESEARCH ARTICLE

© All Rights Reserved by “Journals Club & Co.” 99

Stability Indicating HPLC Method Development for Estimation of Montelukast

Sodium and Acebrophylline in Combined Dosage Form Thesia DU1, Patel BP1

1S. J. Thakkar College of Pharmacy, Avadh Road, Kalawad Road, Rajkot, Gujarat

ABSTRACT Analysis of pharmaceutical product is very important as it concerned with life. Combination of Montelukast sodium and Acebrophylline is used in bronchial asthama and allergic rhinitis. In this Research work, Montelukast sodium and Acebrophylline stock solution was subjected to acid and alkali hydrolysis, oxidation, thermal photolytic and thermal degradation. In this Stability-Indicating method sample was analyzed by reverse phase C18 column (Hibar Lichrospher® 100, RP-18e 5 μm, 250 mm L × 4.6 mm diameter in size) as stationary phase and Acetonitrile:Methanol (60:40 %v/v, pH 3.2 adjusted with O-phosphoric acid) as a mobile phase at a flow rate of 0.8ml/min. Quantification was achieved at 260 nm with PDA detector. Method was validated according to ICH Q2 R1 guideline. The retention time for Montelukast sodium and Acebrophylline was found to be 15.49 minute and 3.45 minute, respectively. The linearity for Montelukast sodium and Acebrophylline was obtained in the concentration range of 5-25 µg/ml and 100-500 µg/ml with mean accuracies of 99.49-100.81% and 99.45-100.51% respectively. Values of %RSD for Precision Study and Robustness was found < 2%. % label claim was found to be 99.23% for MTKT and 100.83% for ACBR. The developed method meets all the acceptance criteria for the validation of analytical method as per the ICH Q2 R1 guideline. The degraded product peaks were well resolved from the pure drug peak with significant difference in their retention-time values. A simple, precise and accurate stability indicating RP-HPLC method was developed for estimation of Montelukast sodium and Acebrophylline in combined Dosage form.

KEYWORDS HPLC, Montelukast sodium, Montelukast, Acebrophylline, Stability Indicating HPLC, Degradation study, ACBR, MTKT

INTRODUCTION

Montelukast sodium (MTKT) 1-[[[(1R)-1-

[3-[(1E)-2-(7-Chloro-2-quinolinyl)

ethenyl] phenyl]-3-[2-(1 hydroxy-1-

methylethyl) phenyl] propyl] thio] methyl]

cyclopropaneacetic acid (Trade name:

Singulair, Montair, Emlucast, Montek,

Montelast, Monti) is a selective and orally

active leukotriene receptor antagonist that

*Address for Correspondence: Purav Talaviya, S. J. Thakkar College of Pharmacy, Avadh Road, Rajkot, Gujarat. Email ID: [email protected]

mailto:[email protected]


inhibits the Cysteinyl leukotriene type-1

receptor (CysLT1) and blocks the action

of Leukotriene D4 (and secondary ligands

LTC4 and LTE4) on the Cysteinyl

leukotriene receptor CysLT1 in the lungs

and bronchial tubes by binding to it. It is

used for the treatment of asthma and to

relieve symptoms of seasonal allergies.1-3

Acebrophylline (ACBR) 1,2,3,6-

tetrahydro- 1,3-dimethyl-2,6-dioxo-7H-

Purine-7-aceticacid compound with trans-

4-[[(2-amino-3,5 dibromophenyl) methyl]

amino] cyclohexanol (Trade name:

cebofyl, AB phylline) is used in bronchial

asthma and pulmonary diseases. It

contains Ambroxol and Theophylline-7-

acetic acid, the former facilitates the

biosynthesis of pulmonary surfactant

while later raises blood levels of

ambroxol, by stimulating surfactant

production. By deviating phosphatidyl

choline towards surfactant synthesis,

making it no longer available for the

synthesis of inflammatory mediators such

as the leukotrienes, acebrophylline also

exerts an inflammatory effect.

Acebrophylline is obtained by targeted

salification of the Ambroxol base and

Theophylline-7-acetic acid.4

This combination is launched by Alkem

Healthcare Ltd. in market as brand name

Abrofyl-M.

Though individual estimation of

Montelukast sodium was done by HPLC,

Voltammetric, Spectrophotometric

methods5-19 and Acebrophylline

estimation was done by HPLC, HPTLC

spectrophotometric

methods.20-24 No

method is available for the estimation of

Montelukast sodium and Acebrophylline

in their combined dosage forms. So it is

thought of interest to develop and validate

chromatographic method for estimation of

these drugs in presence of degradation

products in combined dosage form.

In proposed method, both the drugs are

applied for forced degradation in acid,

alkaline, oxidative, photolytic and thermal

environment and these solutions were

analysed by RP-HPLC method. Stress

study was performed according to ICH

guidelines for stability testing. Method

was developed and validated according to

ICH guidelines.

After systematic and detailed study, the

following procedures are recommended

for the determination of Montelukast

sodium and Acebrophylline in

pharmaceutical formulations.

MATERIALS & METHODS

Montelukast sodium working standard

grade was supplied by Torrent Research

Centre, Ahmedabad. Acebrophylline


working standard grade was supplied by

Ami Life science, Baroda. Acetonitrile,

methanol, water for Chromatography -

Lichrosolv® (Merck Specialities Pvt. Ltd.,

Mumbai), Ortho phosphoric acid 88% GR

(Merck Specialities Pvt. Ltd., Mumbai),

Hydrogen peroxide (30%), LR grade

(Merck, India), Sodium hydroxide AR

grade (Merck specialties Pvt ltd,

Mumbai), Hydrochloric acid 35% pure

AR (Merck specialities Pvt. Ltd.,

Mumbai), Sartorius Filter Paper 0.45

micron (Sartorius, Germany) and Abrofyl-

M tablet formulation-Each tablet contains

10 mg Montelukast sodium and 200 mg

Acebrophylline manufactured by Alkem

Laboratories was purchased from local

market. Identification of both the drugs

were done by interpreting the IR spectra

of pure API of drug sample.

HPLC Instrumentation and Conditions

The HPLC system consisted YL-Clarity

9100 HPLC System (YOUNG-LIN

INSTRUMENT, The Republic of Korea),

column heater and PDA detector (Waters

2998). Data collection and analysis was

performed using YL- Clarity software.

Separation was achieved on Hibar

Lichrospher® 100, RP-18e (5 μm), (250 ×

4.6mm) columns maintained at 25oC using

column oven. Isocratic elution with

Methanol:Acetonitrile (40:60 %v/v), pH

adjusted to 3.2 with O-phosphoric acid at

a flow rate of 0.8 mL/min was carried out.

The detection was monitored at 260 nm

and injection volume was 20 μL. The peak

purity was checked with the photodiode

array detector (PDA).

Preparation of standard solutions and

calibrations

Standard stock solution of ACBR (2000

μg/mL) and MTKT (100 μg/mL) were

prepared in methanol. To study the

linearity range, serial dilutions of ACBR

and MTKT were prepared from 100-500

μg/mL and 5-25 μg/mL in methanol and

injected on to column. For the

construction of calibration curves, five

standard solutions in concentration range

mentioned above were prepared and

injected on to column. Calibration curves

were prepared as concentration of drugs

versus peak area response. The system

suitability test was carried out from six

replicates of standard solution of both the

drug containing 100 μg/mL of ACBR and

5 μg/mL MTKT.

System Suitability Test (SST) and

Analysis of Formulations

The SST ensures the validating power of

the analytical method as well as confirms

the resolution between different peaks of

interest. All critical parameters tested met

the acceptance criteria on all days.

Adequate resolution of ACBR and MTKT

peaks ensured the specificity of the


method. The system suitability assessment

for the analytical HPLC method

established instrument performance

parameters such as peak area, %RSD,

Theoretical Plates (N) and Tailing factor

(Tf) for both the analytes. For analysis of

marketed formulations quantity of powder

from 20 tablets equivalent to 300 mg of

ACBR or 15 mg MTKT were weighed

and transferred to a 100 mL volumetric

flask containing about 20 mL of methanol,

ultrasonicated for 5 min and solution was

filtered through Sartorius Filter paper No.

45 into a 100 mL volumetric flask. Filter

paper was washed with the solvent and

volume was made up to mark. The

solution was suitably diluted with

methanol to get a concentration of 300

μg/mL of ACBR and 15μg/mL of MTKT.

The sample solution was then filtered and

20 μL of the test solution was injected and

chromatogram was recorded for the same

and the amounts of the drugs were

calculated.

Analytical Method Validation

The stability indicating RP-HPLC method

was validated in terms of precision,

accuracy, specificity, sensitivity,

robustness and linearity according to ICH

guidelines. Method precision (inter-day

and intraday) was determined using three

concentrations and three replicates of each

concentration. Standard solutions

containing 100, 300 and 500 μg/mL of

ACBR and 5, 15 and 25 μg/mL of MTKT

were used for precision study.

Repeatability study was determined by

taking six replicates of ACBR (300

μg/mL) and MTKT (15 μg/mL). Assay

method was evaluated with the recovery

of the standards from excipients. Three

different quantities (80%, 100% and

120%) of the standards were added to pre

analyzed formulation and were analyzed

using the developed HPLC method.

Values of Limit of Detection (LOD) and

Limit of Quantitation (LOQ) were

calculated by using σ (standard deviation

of response) and s (slope of the calibration

curve) and by using equations, LOD =

(3.3 x σ)/s and LOQ = (10 x σ)/s. To

determine the robustness of the method,

the final experimental conditions were

intentionally altered and the results were

examined by changing one factor at the

time. The parameters considered (±

values) for the robustness study, flow rate

(± 0.1 mL/min.) and pH (± 1) were

studied.

Degradation study of API and Tablet formulations ACBR and MTKT were subjected to

various forced degradation conditions

individually to effect partial degradation

of the drug preferably in 10-20% range.

The forced degradation study was

performed for the drug product ACBR and


MTKT bulk to determine whether any

observed degradation occurred because of

drug properties or was due to drug

excipients interactions. Forced

degradation of the drug product was

carried out under thermal, photolytic,

acid/alkaline and oxidative stress

conditions. For photolytic stress, drug

product in the solid state was exposed

with UV radiation. Minimum desired

exposure (200 Wh/m2) was observed after

irradiation for 24 h. Sample solution

containing 2000 μg/mL of ACBR and 100

μg/mL were subjected to selected stressed

conditions. Samples except for photo

oxidation were protected from light. For

Acid and base degradation, solutions

containing ACBR and MTKT of the drug

were prepared in 0.1N HCl and 1N NaOH

analysed after 2 h exposure. Oxidative

degradation solution was prepared in

water containing 3% v/v of H2O2 and

analysed after 4 h. Sample for photolytic

studies were exposed UV radiations for 24

h and were used. During dry heat study

samples were exposed to 800C for 24 h in

oven and analysed. After exposure to

desired stress degradation condition,

samples were diluted with methanol to

achieve the nominal concentration of 300

μg/mL of ACBR and 15 μg/mL of MTKT

which were based on their label strength

in standard solution. Formulation

containing ACBR and MTKT were

exposed to same stress conditions and

stress degraded samples were analysed by

following above procedure described for

bulk analytes.

RESULTS AND DISCUSSION

Optimization of Chromatographic condition A well-defined symmetrical peak was

obtained upon measuring the response of

eluent under the optimized conditions

after thorough experimental trials that can

be summarized. The mobile phase was

selected on the basis of best separation,

peak symmetry, theoretical plate etc. A

number of trials were taken for the

selection of mobile phase as mentioned

here. Initially Methanol and Water were

tried in different ratios but result achieved

was not satisfactory. After that Methanol

and Acetoitrile as well as Acetonitrile and

water were tried in different ratio and

again result was found non-satisfactory.

Finally mobile phase consisting of

Acetonitrile and Methanol in the ratio of

60:40% v/v gave good separation and

resolution. The effect of pH (adjusted with

O-phosphoric acid) was studied initially at

higher pH values and it shows baseline

disturbance and peak tailing. pH 3.2 was

found optimum after few of trial and

errors. The effect of various flow rates on

the formation and separation of peaks of

the analytes was studied and a flow rate,

0.8 ml/min was optimum with reasonable


time of analysis. UV detector response of

ACB was studied and the best wavelength

was found to be 260 nm showing highest

sensitivity. Development studies revealed

that methanol: acetonitrile (40:60 %v/v)

pH adjusted to 3.2 with O-phosphoric acid

at a flow rate of 0.8 ml/min was suitable

conditions for a stability indicating

method. ACBR was having retention time

3.45 min and MTKT was having retention

time 15.49 min. degraded products of

ACBR and MTKT were well separated.

Analytical Method Validation

The method was validated according to

ICH guidelines. The following validation

characteristics were addressed: linearity,

range, accuracy, precision, sensitivity

(LOQ and LOD) and robustness.

Specificity of the method was determined

by analyzing solutions containing drug

product, excipients and stress degraded

samples. All chromatograms were

examined to determine if ACBR and

MTKT and its stress degraded product

coeluted with each other or with any

excipient peak. Peak purity of stressed

samples of ACBR and MTKT were

checked by using PDA detector. The

purity angle within the purity threshold

limit obtained in all stressed samples

demonstrated the analyte peak

homogeneity.

Linearity, Range, LOD and LOQ

The linearity range for MTKT and ACBR

was found to be in the range of 5-25 μg/ml

and 100-500 μg/ml respectively.

Calibration data for MTKT and ACBR is

presented in table 1 and 2. Overlay

Chromatogram of Std. API mixture of

both the drugs is shown in fig.

1.Calibration curve of both the drugs are

shown in fig. 2 and 3.

System Suitability Test (SST)

SST results are presented in Table 3.

Chromatogram for system suitability is

shown in fig. 4.

Accuracy

The data for accuracy for MTKT and

ACBR are presented in table 4 and 5

respectively. The recovery range for

MTKT and ACBR were found to be

99.49-100.81% and 99.45-100.51%

respectively.

Precision

Repeatability

The data for repeatability of MTKT and

ACBR is depicted in table 6. The %RSD

was found to be 0.511% and 0.863% for

MTKT and ACBR respectively.

Intraday precision

The data for intraday precision of MTKT

and ACBR are presented in table 7. Range

of %RSD was found to be 0.179-0.701%

for MTKT and 0.142-0.586% for ACBR.

Interday precision


The data for interday precision of MTKT

and ACBR are summarized in table 8.

Range of %RSD was found to be 0.077-

0.391% for MTKT and 0.088-0.383% for

ACBR.

Robustness

The data for robustness for MTKT and

ACBR are presented in table 9.

Robustness of the method was evaluated

by i) change in flow rate ii) change in pH.

Assay of formulation Formulation was procured commercially

from the market. Formulation was

analyzed for simultaneous estimation of

MTKT and ACBR by the RP-HPLC

method. The assay values for MTKT and

ACBR are presented in Table 10. The

result of dosage form analysis by

developed method was compatible with

the labelled amount of each component of

tablet.

Stability Indicating Study Analytes and its stress degradation

product were well separated. Although the

conditions used for forced degradation

were attenuated to achieve degradation in

the range 10–30%. The drug was

extensively degraded by acid hydrolysis,

alkaline hydrolysis, photolytic, Thermal

and oxidative condition. Chromatograms

of acid, alkali, oxidative, Photolytic and

Thermal degradation of ACBR and

MTKT in combined formulation are

shown in Fig. 5 to 9, respectively. Stress

conditions used and are presented in Table

11 and 12. Chromatographic peak purity

data was obtained from the spectral

analysis report

Figures and Tables

Fig. 1 : Overlay chromatogram of Std. API mixture of MTKT and ACBR


Fig. 2 : Calibration curve of MTKT

Fig. 3: Calibration curve of ACBR

Fig. 4: Chromatogram of Standard MTKT (5 μg/ml) and ACBR (100 μg/ml)


Fig. 5 : Degradation peak of Standard API mixture of ACBR and MTKT in 0.1 M HCl

after 2 hrs.

Fig. 6 : Degradation peak of Standard API mixture of ACBR and MTKT in 1M NaOH after 2 hrs

Fig. 7 : Degradation Peak of std. API mixture of ACBR and MTKT in 3%v/v H2O2 after 4 hrs.


Fig. 8 : Degradation peak of std API mixture of ACBR and MTKT after 24 hrs of UV exposure

Fig 9 : Degradation peak of std API mixture of ACBR and MTKT after 24 hrs. in Hot air oven for 80oC

Table 1: Data of Linearity

Sr. No.

Conc. in μg/ml Area ± SD

MTKT ACBR MTKT ACBR

1 5 100 1109.784 ± 7.3970 1143.628 ± 3.6021

2 10 200 2331.780 ± 6.1427 2541.217 ± 5.0723

3 15 300 3445.345 ± 4.0549 3462.522 ± 5.8670

4 20 400 4715.748 ± 5.7981 4593.376 ± 6.9692

5 25 500 6016.050 ± 8.5052 5837.654 ± 6.4161

Correlation co-efficient 0.999 0.999

Slope 243.9 11.64

Intercept 135.2 16.66

Regression equation 243.9x - 135.2 11.64x - 16.66


Table 2: Data of LOD and LOQ

Table 3 : Data of System suitability parameters

Sr. No.

Standard Response (mV*S) Std. Value

MTKT ACBR 1 1109.784 1143.628

2 1118.458 1151.912 3 1114.298 1131.324 4 1102.259 1147.584 5 1121.655 1160.235

Average 1113.262 1146.935 SD 1.8017 1.1800

%RSD 0.4871 0.0590 ≤ 2 % Retention time 16.49 3.450

Theoretical plates 5056 10303 > 2000 Tailing Factor 1.359 1.568 Not more than 2

Resolution 2.180 > 2

Table 4 : Accuracy data of MTKT

Spiked level (%)

Conc.

in sample (μg/ml)

Conc. added

in (μg/ml)

Total conc.

(μg/ml)

Conc. Recovered

% Recovery

SD

%RSD

80%

10 8 18 18.17 100.94 0.2218

0.2210 10 8 18 18.12 100.67

10 8 18 18.09 100.50 Avg. 100.70

100%

10 10 20 20.16 100.80 0.1258 0.1248 10 10 20 20.19 100.95

10 10 20 20.14 100.70 Avg. 100.81

120%

10 12 22 21.86 99.35 0.1450

0.1457 10 12 22 21.89 99.50

10 12 22 21.92 99.64 Avg. 99.49

MTKT(μg/ml ) ACBR(μg/ml )

LOD 0.4807 1.412

LOQ 1.4570 4.281


Table 5 : Accuracy data of ACBR

Spiked

level (%)

Conc.

in sample (μg/ml

Conc. added

in (μg/ml)

Total conc.

(μg/ml)

Conc. Recovered

% Recovery

SD

%RSD

80%

200 160 360 361.89 100.52

0.0251

0.0250 200 160 360 361.94 100.54

200 160 360 361.77 100.49 Avg. 100.51

100% 200 200 400 397.51 99.38

0.0750

0.0755 200 200 400 398.13 99.53

200 200 400 397.85 99.46 Avg. 99.45

120% 200 240 440 442.32 100.53

0.0568

0.0566 200 240 440 441.98 100.45

200 240 440 441.86 100.42

Avg. 100.46

Table 6 : Data of Repeatability study

Sr No.

Peak area at 260 nm (mV*s)

MTKT ACBR

1 3445.345 3462.522

2 3421.257 3445.147

3 3455.236 3489.371

4 3425.347 3411.482

5 3467.589 3465.548

6 3440.951 3476.193

Mean 3442.620 3458.377

S.D. 17.59291 27.27936

% RSD 0.511 0.863


Table 7 : Data of Intraday Precision

Conc. (μg/ml)

Area Mean ± % SD

(mV*s)

%RSD

Area Mean ± % SD

(mV*s)

%RSD

MTKT ACBR MTKT ACBR

5 100 1110.835 ± 7.7917 0.7015 1147.62± 6.7337 0.5863

15 300 3495.249± 6.2657 0.1795 3505.40± 5.0075 0.1426

25 500 5962.317± 11.0949 0.1859 5803.62± 8.9770 0.1547

Avg. 0.3014 Avg. 0.2945

Table 8 : Data of Interday Precision

Conc. (μg/ml)

Area Mean ± % SD

(mV*s)

%RSD

Area Mean ± % SD

(mV*s)

%RSD

MTKT ACBR MTKT ACBR

5 100 1112.546± 4.3711 0.3912 1145.32± 4.4734 0.3891

15 300 3497.478± 3.0412 0.08702 3508.12± 7.0500 0.2009

25 500 5959.987± 4.6282 0.07761 5801.99± 5.1117 0.0880

Avg. 0.1852 Avg. 0.226

Table 9: Data for Robustness study

Robustness parameter

Drug

Rt(min.) ± SD

%RSD

Change in flow rate

0.9 ± 0.1ml/min. MTKT 15.60 ± 0.0551 0.5314

ACBR 3.41 ± 0.0300 0.2327

1.0 ± 0.1ml/min. MTKT 15.59 ± 0.0252 0.2561

ACBR 3.44 ± 0.0550 0.3097

Change in pH

3.5 MTKT 15.66 ± 0.0351 0.3349

ACBR 3.39 ± 0.0569 0.4397

4 MTKT 15.64 ± 0.0473 0.4835

ACBR 3.47 ± 0.0153 0.1276


Table 10 : Data for Analysis of marketed formulation

Amount taken (mg)

Average amount found (mg) ± SD % Label claim ± SD

MTKT 15 14.88 99.23 ± 1.1229

ACBR 300 302.5 100.83 ± 0.2544

Table 11 : Data of Degradation study of MTKT

Degradation condition

Time (hrs) Area Conc.

(μg/ml) % Assay % Degradation

Retention Time

Acidic/ 0.1 M HCl /2 hr./ Solution

0 3449.22 14.92 99.46 12.60 2.91

2 415.94 1.79 87.40 Alkaline/1.0 M

NaOH/RT/ 2 hr/ Solution

0 3441.88 14.90 99.33 24.45 6.22

2 825.884 3.57 75.75 Peroxide/ 3%

H2O2/4 hr/ Solution

0 3446.75 14.93 99.53 19.28 7.30

2 654.74 2.83 80.72 Photo/under UV

ligtht / 24 hr/Solid

0 3439.32 14.86 99.06 10.61 2.89

24 343.89 1.49 89.39

Thermal / 80°C/ 24 hr/ Solid

0 3440.02 14.88 99.20 3.60 6.78 24 123.84 0.54 96.40

Table 12 : Data of Degradation study of ACBR

Degradation condition

Time (hrs) Area Conc.

(μg/ml) % Assay % Degradation

Retention Time

Acidic/ 0.1 M HCl /2 hr./ Solution

0 3468.54 301.23 100.41 13.65 14.98 2 471.64 39.16 86.35

Alkaline/1.0 M NaOH/RT/

2 hr/ Solution

0 3465.23 300.12 100.04 18.89 13.95

2 658.35 57.02 81.11 Peroxide/ 3%

H2O2/4 hr/ Solution

0 3470.15 300.21 100.07 21.66 14.09

2 728.70 63.04 78.34 Photo/under UV

ligtht / 24 hr/Solid

0 3462.65 299.72 99.90 11.65 6.96

24 3482.12 29.97 88.35

Thermal / 80°C/ 24 hr/ Solid

0 3466.25 299.23 99.74 6.95 13.92 24 242.62 20.94 93.05


CONCLUSION

Stability-indicating RP-HPLC method for

estimation of Montelukast sodium and

Acebrophylline in their solid dosage form

was established and validated as per the

ICH guidelines. Different degradation

products were found for drug substance

and drug product in acidic, alkaline,

oxidative, thermal and photolytic

degradation. Peak of degraded products

were not interfering with that of main

drug. The developed method is simple,

precise, accurate, specific, and robust.

Hence it can be used for the routine

analysis of Montelukast sodium and

Acebrophylline. Further this present

method could be helpful for the

identification of impurities through LC-

MS, degradation kinetic studies and

establishment of probable degradation

pathway.

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HOW TO CITE THIS ARTICLE

Thesia, D. U., Patel, B. P. (2014). Stability Indicating HPLC Method Development for Estimation of Montelukast Sodium and Acebrophylline in Combined Dosage Form Journal Club for Pharmaceutical Sciences (JCPS), 1(I), 99-114

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Stability Indicating HPLC Method Development for Estimation of Montelukast Sodium and Acebrophylline in Combined Dosage Form